U.S. patent application number 13/514046 was filed with the patent office on 2012-10-25 for standardization of tissue specimen preservation by ultrasound and temperature control.
Invention is credited to Wei-Sing Chu.
Application Number | 20120270293 13/514046 |
Document ID | / |
Family ID | 44146112 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120270293 |
Kind Code |
A1 |
Chu; Wei-Sing |
October 25, 2012 |
STANDARDIZATION OF TISSUE SPECIMEN PRESERVATION BY ULTRASOUND AND
TEMPERATURE CONTROL
Abstract
A tissue sample preservation process and device for improving
and standardizing tissue preservation procedure, including placing
tissue specimens in a cold fixative, performing fixative
penetration at a refrigerated temperature, and accelerating
fixative penetration by ultrasound. Also disclosed is the
application of ultrasound and temperature control in the
dehydration, clearing, and impregnation steps.
Inventors: |
Chu; Wei-Sing; (Silver
Spring, MD) |
Family ID: |
44146112 |
Appl. No.: |
13/514046 |
Filed: |
December 1, 2010 |
PCT Filed: |
December 1, 2010 |
PCT NO: |
PCT/US10/58521 |
371 Date: |
June 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61283558 |
Dec 7, 2009 |
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Current U.S.
Class: |
435/173.5 ;
435/307.1; 435/374 |
Current CPC
Class: |
A01N 1/0278 20130101;
A01N 1/0284 20130101 |
Class at
Publication: |
435/173.5 ;
435/374; 435/307.1 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12M 1/42 20060101 C12M001/42; C12N 5/07 20100101
C12N005/07 |
Claims
1. A method of preserving tissue specimens comprising: a)
submerging said tissue specimens into a fixative of a temperature
between 0 to 25 degree C., b) subjecting said tissue specimens and
said fixative to a cooling mechanism, and, c) subjecting said
tissue specimens and said fixative to a convection mechanism.
2. The method of claim 1, wherein said fixative is a formaldehyde
water solution comprising 1% to 10% of formaldehyde by weight, and
wherein said temperature is between 4 to 20 degree C., preferably
between 4 to 18 degree C.
3. The method of claim 1, wherein said fixative is a solution
comprising at least 50% by volume of at least one chemical from the
following group: ethanol, methanol, PEG, and acetone; and wherein
said temperature is between 4 to 25 degree C., preferably between 4
to 18 degree C.
4. The method of claim 1, wherein said convection mechanism is
ultrasound irradiation.
5. The method of claim 4, wherein said ultrasound is of a frequency
between 0.1 MHz to 5 MHz, preferably between 0.2 MHz to 2 MHz, and
further preferably between 0.4 MHz to 1.5 MHz.
6. The method of claim 5, wherein said ultrasound is of a power
between 1 to 100 Watts, preferably between 5 to 50 Watts, and
further preferably between 10 to 30 Watts.
7. A method of claim 6 further comprising at least one dehydration
step of submerging fixed tissue specimens in a dehydration solution
of a temperature between 0-25 degree C.
8. A method of claim 6, further comprising: d) turning off said
cooling mechanism after a predetermined length of time; e)
continuing ultrasound irradiation to increase temperature of said
tissue specimens in said fixative to a predetermined value; f)
switching ultrasound irradiation on and off to maintain said
predetermined temperature value for a predetermined length of
time.
9. A method of claim 6 further comprising: at least one dehydration
step; comprising: i) submerging fixed tissue specimen in a
dehydration solution of a temperature between 0-40 degree C., ii)
raising the temperature of said dehydration solution to a
predetermined value by ultrasound irradiation alone or in
combination with other heating means, and, iii) keeping the
temperature value for a predetermined length of time; at least one
clearing step, comprising: i) submerging dehydrated tissue specimen
in a clearing solution, ii) raising the temperature of said
clearing solution to a predetermined value by ultrasound
irradiation alone or in combination with other heating means, and,
iii) keeping the temperature for a predetermined length of time: an
impregnating step, comprising: i) submerging the dehydrated and
cleared tissue specimen in melted wax, ii) irradiating said
dehydrated and cleared tissue specimen in melted wax with
ultrasound for a predetermined length of time, and, iv) applying a
vacuum to said dehydrated and cleared tissue specimen in melted
wax.
10. The method of claim 1, wherein said convection mechanism is by
stirring, agitation, or pumping-in and pumping-out.
11. The method of claim 1, further comprising: d) subjecting said
tissue specimens and said fixative to low pressure, wherein said
low pressure is in the range of 10 to 20 inch Hg.
12. The method of claim 1, further comprising: d) subjecting said
tissue specimens and said fixative to high pressure, wherein said
high pressure is a pressure that is above atmospheric air
pressure.
13. The method of claim 1, further comprising: d) subjecting said
tissue specimens and said fixative to alternative high pressure and
low pressure.
14. A device for preserving tissue specimens in an ultrasound
field, comprising: 1) At least one vessel for holding tissue
specimens and preservation reagent(s), 2) An ultrasound generating
system to produce ultrasound field in reagent(s), and 3) A
temperature control system for maintaining a preset temperature in
said vessel.
15. A device of claim 14, wherein said ultrasound generating system
produces ultrasound signal of: 1) a frequency in the range of 0.1
MHz and 5 MHz, preferably in the range of 0.2 MHz and 2 MHz,
further preferably in the range of 0.4 MHz and 1.5 MHz; and, 2) a
power in the range of 0.1 to 100 Watts, preferably between 5 to 50
Watts, further preferably between 10 to 30 Watts.
16. A device of claim 15, wherein said ultrasound generating system
comprises at least one piece of piezoelectric transducer, said
piezoelectric transducer can be in the shape of a circular disc, a
rectangle bar, a cylinder, or an array of multiple transducers.
17. A device of claim 16, wherein said piezoelectric transducer(s)
is attached to the surface of the wall or the bottom of said vessel
by a glue. 26
18. A device of claim 16, wherein said piezoelectric transducer (s)
is sealed in a transducer housing.
19. A device of claim 18, wherein said transducer housing is
submerged in said tissue preservation reagent(s).
20. A device of claim 18, wherein ultrasound is produced from one
side of said transducer housing to irradiate on tissue specimens
that are placed on the same side of said transducer housing
emitting ultrasound.
21. A device of claim 18, wherein ultrasound is produced from both
sides of said transducer housing to irradiate on tissue specimens
that are placed on both sides of said transducer housing.
22. A device of claim 15, wherein said ultrasound generating system
can deliver continuous or pulsed ultrasound field with fixed or
sweep frequency(s).
23. A device of claim 14, wherein said vessel contains a side wall,
a detachable cover, and a movable bottom fitted water tight to the
side wall; said movable bottom is connected with a revolving
engine; said revolving engine drives back and forth movements of
said moveable bottom.
24. A device of claim 14, wherein said vessel contains a side wall,
a bottom, and a movable cover; said movable cover is fitted water
tight to the side wall and is connected with a revolving engine;
said revolving engine drives back and forth movements of said
movable cover.
25. A device of claim 14, wherein said temperature control system
contains a cooling means and a heating means.
26. A device of claim 24, wherein said cooling means is circulating
a cold liquid through a space surrounding said vessel; said space
is formed between the outside surface of said vessel and a shell
surrounding said outside surface of said vessel; and said cold
liquid is of a temperature between -20 degree to 25 degree C.
27. A device of claim 24, wherein said cooling means is circulating
a cold liquid through a pipe passing through said tissue
preservation reagent.
28. A device of claim 24, wherein said cooling means is a
semiconductor device.
29. A device of claim 24, wherein said heating means is ultrasound
irradiation alone or in combination of ohmic heating or microwave
heating.
Description
BACKGROUND OF INVENTION
[0001] 1. An Overview of the Field of Tissue Preservation
[0002] Immediately after a tissue specimen is removed from patient
body, the ischemic cascade begins. The ischemic cascade leads to
changes of many susceptible cellular biomolecules such as mRNA
degradation and protein dephosphorylation in the tissue specimen.
Therefore the longer the ischemia, the more pre-analytical changes
will happen in the patient tissue specimens: Such changes often
hamper correct and sensitive molecular diagnosis and prognosis
assays [Liotta 2000, Emmert-Buck 2000, Compton 2007, Hewitt 2008,
Espina 2008]. Flash-freezing by which tissue specimens are
preserved in a deep frozen state to prevent biomolecule changes, is
a standard protocol to preserve tissue specimens for molecular
analysis. However, in addition to the high cost and sophistication
of the equipment, flash-freezing destroys the morphology of the
tissue specimens. Good tissue morphologies are paramount
requirements for diagnostic tissue samples.
[0003] In the current practice of surgical histology, tissue
specimens are placed, bulk or grossed, into a fixative for
fixation. Fixed tissues are then processed through dehydration,
clearing, paraffin impregnation, and embedded in a paraffin block.
Formalin is the most commonly used fixative by the clinical
pathology community [Hewiit 2008, Fox 1985, 1987, Boon 1988].
Modern histology is based on the tissue morphologies produced by
the formalin fixed and paraffin embedded (FFPE) tissues. Formalin
fixation is carried out at room temperature overnight or longer in
most clinical pathology laboratories. Susceptible biomolecules may
degrade before formalin fully diffuses into the specimen. Reactions
between formaldehyde and proteins happen quickly at room
temperature, causing extensive cross-linking formation in the
tissue periphery and little cross-linking formation in the tissue
center. Prolonged fixation time is sometimes required for the
center area to catch up with peripheral area in the extent of
cross-linking [Medawar 1941; Boon 1988; Helander 1994, 1999;
Ruijter 1997].
[0004] The formalin fixed tissue samples have the following 2 major
problems: (1) biomolecules are heavily modified by extensive
cross-linking; and, (2) due to varied fixation times, cross-linking
levels vary in different samples. Therefore conventional FFPE
tissue samples are generally not standardized. A lack of
standardization and heavy modification of biomolecules are the
major obstacles preventing FFPE tissue samples from being used in
quantitative molecular analysis.
[0005] A number of none cross-linking fixatives are developed in
attempts to accommodate quantitative molecular analysis [Wenk 2006;
Wester K 2003; Boon 2008; Espina 2009]. Tissue morphologies
produced by non cross-linking fixatives are different from those
produced by formalin--a major factor hampering wide acceptance of
non cross-linking fixatives in the clinical pathology community. An
ideal solution for tissue preservation problems is to develop a
method that can generate both the gold standard FFPE morphology and
high quality biomolecules, comparable to those from flash-frozen
tissues, in a simple and cost effective way.
[0006] 2. Description of the Related Art
[0007] Conventional methods prepare tissues for histology by
incubation in separate solutions of phosphate-buffered 10%
formaldehyde for fixation, a series of increasing concentrations of
ethanol for dehydration, and xylene for clearing tissue of
dehydration agent, prior to impregnation with paraffin. Because of
the time required for this process, usually 8 hours or longer, it
is customary to complete these separate steps--fixation,
dehydration, clearing, and impregnation--overnight in automated
mechanical instruments designed for those tasks (see, for example,
U.S. Pat. Nos. 3,892,197, 4,141,312, and 5,049,510). A typical
automated tissue processor (TISSUE-TEK) requires more than eight
hours and is programmed to process batches of tissue samples.
[0008] In water solution, formaldehyde molecules [HCOH] is hydrated
to form methylene glycol [CH.sub.2(OH).sub.2] which exists at
equilibrium with unhydrated formaldehyde molecules, as shown
below:
HCOH+H.sub.2OCH.sub.2(OH).sub.2
[0009] Low temperature tilts the equilibrium to methylene glycol
molecules which penetrate tissues much faster than unhydrated
formaldehyde molecules. However, it is the formaldehyde molecules
that form the cross-linking bridge. There is a conundrum in the
formalin fixation in relation with temperatures: 1) Low temperature
favors biomolecule preservation in tissue specimens, slows down
cross-linking, facilitates even diffusion of formalin molecules
throughout tissue specimens; 2) High temperature favors HCOH
formation which in turn facilitated cross-linking, while
cross-linking of proteins in tissue periphery slows down formalin
penetration into the tissue center; 3) At a low temperature speed
of molecule diffusion in general is low due to reduction in
molecule motion and leads to significant reduction in penetration
speed for formalin molecules.
[0010] At room temperature, the sequence of the events for formalin
fixation of a routine tissue specimen (1-4 mm in thickness) is as
follows: (1) Rapid diffusion of methylene glycol to reach the
interior requires approximately 1-4 h; (2) The succeeding steps
(dehydration of methylene glycol and cross-linking reaction)
together require approximately 24 h. Therefore, diffusion of
methylene glycol in this case is not hindered by a dense network of
cross-linked proteins. However, at high temperatures, all three
steps are accelerated. Because dehydration and cross-linking also
are completed first in the parts of the heated tissue where
methylene glycol is present, i.e., the periphery of the tissue
block, further diffusion into the center is hindered by the dense
protein network thus created.
[0011] To accelerate tissue processing, U.S. Pat. Nos. 4,656,047,
4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos.
3,961,097, 5,089,288, and 6,291,180 use ultrasonic energy; and U.S.
Pat. No. 5,023,187 uses infrared energy.
[0012] Three factors of ultrasound that can influence tissue
fixation are as following: 1) microcavitations and highly efficient
convection produced by ultrasound in the fixative and the tissue
samples increase tissue permeability to fixative molecules; 2)
ultrasound causes gradual temperature increase leading to gradual
increase in cross-linking speed; 3) ultrasound may generate free
radicals in the fixative which also favor cross-linking
formation.
[0013] As the personalized medicine is looming, there is an urgent
need for high quality and standardized tissue specimens. This need,
when translated into the FFPE tissue sample preparation, requires
that tissues being fixed quickly and evenly, and processing steps
standardized. Therefore, it is desirable to establish procedures
and possibly instruments to achieve all these.
[0014] Lack of standardization for protocols as well as lack of
on-site quality control in tissue fixation by formalin are among
the major drawbacks for the FFPE tissue samples. The long time
involved in the fixation, especially that of large specimens is a
major limitation in making a timely diagnosis. It is also
considered a major factor affecting the incentives to change the
pre-fixation tissue handling procedures in both the operating rooms
and pathology laboratories. In most hospitals, the tissues are
placed in formalin in the operating room; therefore, the
pathologist cannot control fixation time. Accordingly, it is
virtually impossible to standardize quantitative assays on paraffin
sections, because the duration of formalin fixation influences the
degree of cross-linking which in turn affects the availability of
biomolecules for quantitative assays. In addition, solid specimens
larger than 25 gram are not always completely fixed after being
submerged in formalin for 24 hr. Cutting the surgical specimen into
smaller pieces facilitates fixation, but is undesirable for final
anatomical orientation and often delays pre-fixation time.
[0015] Changes of macromolecule composition, especially cell
components involved in cell signaling pathways, as well as tissue
autolysis, happen immediately after tissue is removed from
patients. In protocols used by most pathological departments
formalin fixation is done at room temperature or even higher to
facilitate formalin penetration. During the prolonged penetration
stage at ambient temperatures, autolytic degradation of susceptible
biomolecules, such as mRNA, phosphorylated proteins, and protein
antigens may occur. In addition to macromolecule changes,
progressive formalin fixation at ambient temperature causes
prolonged exposure to formalin at the periphery, leading to over
fixation, while fixation is incomplete at the center of large
specimens. This uneven fixation often leads to inconsistent results
in immunohistochemistry assays and possibly many other molecular
tests.
SUMMARY OF THE INVENTION
[0016] The invention is about processes and devices that are used
in the fixation and processing of tissue specimens facilitated by
ultrasound and temperature control to achieve standardized tissue
specimen preservation. First of all, the invention relates to a low
temperature fixation procedure which comprises: (a) submerging
tissue specimens in a fixative at a refrigerated cold temperature;
(b) irradiating the tissue specimens in the fixative with
ultrasound with a superimposing cooling system to keep the
temperature of the tissue specimen and fixative low; (c)
optionally, turning off the superimposing cooling system and raise
temperature of the tissue specimens and the fixative by continued
ultrasound irradiation alone or in combination with another heating
means; and, (d) optionally, stopping (quenching) the cross-linking
reaction by chemical reagents or cooling.
[0017] The invention also relates to a tissue specimen preservation
procedure, comprising: (a) a fixation step which comprises: i)
tissue specimens are submerged in fixative immediately after tissue
specimens are excised from patients and kept at cold temperature
until the next step in the process; ii) tissue specimens in cold
fixative are irradiated by ultrasound and the specimen/fixative
temperature is kept low by a cooling system superimposed on the
ultrasound irradiation for thorough and uniform penetration of
fixative molecules; iii) optionally, the fixative temperature is
then raised by ultrasound, microwave, or energy of other kinds,
when the cooling system is turned off; (b) at least one dehydration
step which comprises submerging fixed tissue specimen in a
dehydration solution and irradiating the fixed tissue specimen with
ultrasound; (c) at least one clearing step which comprises
submerging the fixed and dehydrated tissue specimen in a clearing
solution and irradiating the fixed and dehydrated tissue specimen
with ultrasound; (d) an impregnating step which comprises
submerging the fixed, dehydrated, and cleared tissue specimen in
melted wax or paraffin and irradiating the fixed, dehydrated, and
cleared tissue specimen with ultrasound.
[0018] The invention also relates to a tissue specimen preservation
device comprising at least one vessel for holding tissue specimens
and preservation reagents, an ultrasound generating system to
produce ultrasound field in tissue preservation reagents, and a
temperature control system that is coupled with the ultrasound
generating system for maintaining a cold temperature.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Tissue specimen preservation workflow for
standardization of pre-analytical tissue specimen preservation by
ultrasound and temperature control.
[0020] FIG. 2. Cross-linking effect of fixative solutions with
different formaldehyde concentrations.
[0021] FIG. 3. Lysozyme, BSA, myoglobin, RNase A, trypsin, and egg
white were each dissolved (or diluted) in 1.times. PBS, pH 7 at 20
mg/ml. Aliquots of 50 micro liter of each protein solution were
mixed with equal volumes of NBF and incubated at designated
temperatures for 10 minutes. After incubation, 6 micro liter of
each incubation mixture was mixed with 2 micro liter of 4.times.
SDS loading buffer and loaded in a well on SDS PAGE, separated by
electrophoresis, and stained with Coomassie blue.
[0022] FIG. 4. One configuration of US chamber in which US
transducer is included in a transducer housing removable from the
solution chamber (reaction chamber). In this configuration, sound
wave is emitted in one direction. Tissue specimens may be removably
attached to the housing and can be moved together with the housing
from one reaction chamber to the next reaction chamber.
[0023] FIG. 5. One configuration of US chamber in which US
transducer is included in a transducer housing removable from the
solution chamber (reaction chamber). In this configuration, sound
wave is emitted in two directions. Tissue specimens may be
removably attached to the housing and can be moved together with
the housing from one reaction chamber to the next reaction
chamber.
[0024] FIG. 6. One configuration of US chamber coupled with a
cooling/heating system. In this configuration, fixative or
processing solutions are cooled or heated in a cooling/heating
device and circulated in and out of the US chamber.
[0025] FIG. 7. One configuration of a cylinder/piston device to
generate vacuum and pressure in the US chamber. In this
configuration, the bottom part of the US chamber which is affixed
with a US transducer moves up and down to produce pressure and
vacuum in the US chamber.
[0026] FIG. 8. One figuration of a cylinder/piston device to
generate vacuum and pressure in the US chamber. In this
configuration, the top cover of the US chamber moves up and down to
produce pressure and vacuum in the chamber.
[0027] FIG. 9. Comparison of H&E staining of bovine kidney (1),
liver (2), and pancreas (3) tissues fixed at (A) room temperature
overnight and (B) 4.degree. C. (upper panel); and the
quantification of number of nuclei per field as well as nuclear
size (lower panel).
[0028] FIG. 10. H&E staining of center and periphery of liver
tissue fixed with and without US irradiation.
[0029] FIG. 11. IHC and Western blot studies on cow kidney tissue
fixed for 30 min at 4 C with US irradiation (US-LT-FFPE) and for
overnight at room temperature (FFPE). IHC assays against vimentin
and cytokeratin were done without antigen retrieval. Western blot
assays were done with whole protein extracts from same amounts of
tissue samples fixed with respective method.
[0030] FIG. 12. The comparison of inhibition activity to RNase A at
RT by Formalin with, RNA Later, 50% methanol, and in PBS with
autoclave (120.degree. C.) for 30 minutes.
[0031] FIG. 13. RNase A at designated amount was incubated with NBF
at RT or 4.degree. C. for 5 min and then mixed with 2 .mu.g yeast
tRNA and incubated at room temperature for 30 min for RNase A
digestion and separated by 2% agarose gel. The tRNA molecules were
between 50-100 nucleotides in length.
[0032] FIG. 14. SDS PAGE and Western blot analysis on lysates of
T47D cells fixed in neutral buffered formalin at 4 C or RT for
various lengths of time. Left: SDS PAGE stained by Coomassie
Blue.
[0033] Middle: Western blot membrane probed with pooled antibodies
against HER-2 and ER. Right: Plots for digital intensities of HER-2
and ER Western blot signals against fixation time.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Currently, almost all clinical tissue specimens are fixed in
formalin at room temperature [Hewitt 2008, Boon 2008]. Fixing
tissues in cold formalin is considered time consuming and used
occasionally for research purposes. The obstacles for wide
acceptance of the cold formalin fixation procedure in the clinical
community include the following:
[0035] 1. It is commonly believed that an adequate cross-linking
level must be reached in the tissues to be fixed to ensure proper
preservation of microscopic details.
[0036] 2. It takes much longer for tissue specimens to reach the
desired cross-linking level at cold temperature than at room
temperature.
[0037] 3. It is more convenient to fix tissue specimens at room
temperature.
[0038] We have shown that tissue specimens fixed by formalin at 4 C
overnight generated excellent FFPE morphology, although the tissue
specimens still looked fresh, indicating that a marginal level of
cross-linking was sufficient to produce the gold standard formalin
morphology. The integrity and availability of biomarkers in the
cold-formalin-fixed tissues were better than those in tissues fixed
by the conventional room temperature method for
immunohistochemistry (INC) and Western blot analysis. We further
demonstrated that ultrasound (US) irradiation could significantly
reduce time needed in fixation of tissue specimens in formalin at
cold temperature.
[0039] A good tissue specimen preservation strategy should
demonstrate the ability to instantly inhibit autolysis (i.e.,
inactivation of endogenous destructive enzymes), the ability to
minimize biomolecule modifications, the ability to produce
preserved tissue samples of best cellular and subcellular
morphology, and the ability for as much as possible macromolecules
to be recovered from preserved tissue samples.
[0040] Neutral buffered formalin (NBF), often referred to as 10%
NBF, is a 10-fold dilution of saturate formaldehyde solution in
phosphate buffered saline (PBS), and has the formaldehyde
concentration of about 3.7-4%. NBF is a commonly used cross-linking
fixative in routine tissue preservation. Solutions with
formaldehyde concentration lower than 4%, e.g., 2%, 1% and even
lower were tested for their cross-linking effect to proteins. The
lower the formaldehyde concentration is the less effective is the
fixative solution in forming cross-linking or modifications, thus
cross-linking or biomolecule modification level is often low.
[0041] Disclosed here is a method to control and standardize the
cross-linking/modification levels in tissue fixation by lowering
the concentration of fixative agents. Compared with NBF (e.g.,
3.7%-4% formaldehyde), penetration of formaldehyde solution of
lower concentration into tissue specimen is slower. However,
facilitated by ultrasound and/or other means to increase
permeability of tissue specimens, fixative solutions with lower
formaldehyde concentration can be used and less toxic formaldehyde
waste will be generated. Since less cross-linking and other
biomolecule modifications are formed, solutions with lower
formaldehyde concentrations can further facilitate biomolecule
recovery from fixed tissue samples as well as standardization of
tissue fixation by producing a uniform cross-linking/modification
level throughout the tissue specimen.
[0042] Also disclosed is a method to control the
cross-linking/modification level, as well as slowing the tissue
auto degradation, during tissue fixation by low temperature. At a
cold temperature, biomolecule cross-linking/modification by
formaldehyde is slow, so a uniform and moderate level of
cross-linking/modification can be readily achieved throughout the
tissue specimen. Since enzymatic activities are greatly reduced at
cold temperatures, ischemic cascade and tissue degradation will be
greatly reduced. A cold temperature (e.g., 0 degree C. to 25 degree
C., preferably 4 C to 20 C, further preferably 4 C to 18 C) can be
applied throughout fixation step. It can also be applied as a first
penetration phase which is followed by warm cross-linking phase. Or
it can even be applied at a sequence of cold phase-warm phase-cold
phase, where the second cold phase is used to stop or reduce the
cross-linking speed in the warm phase.
[0043] Another method to control and standardize the
cross-linking/modification levels is by addition of quenching
(stopping) reagents to the fixative solutions.
[0044] Further disclosed is a facilitated two-phase procedure for
the fixation step with both cross-linking and non-cross-linking
fixative agents. For cross-linking fixatives, such as formaldehyde,
the invention relates to a penetration phase at cold temperature,
optionally followed by a cross-linking phase at hot or ambient
temperature: at cold temperature, activity of most enzymes in the
ischemic cascade is suppressed; at cold temperature, cross-linking
reaction is suppressed, facilitating even penetration of
formaldehyde molecules throughout tissue specimens; at high
temperature, cross-linking reaction is fast and reaction time can
be limited in a short period facilitating standardization of
cross-linking levels.
[0045] Cross-linking temperature and time can be adjusted to
produce preferred level of cross-linking/modifications. For
example, tissue specimens can be fixed by NBF at 4-10 degree C. for
overnight with or without ultrasonic irradiation, followed by
routine dehydration, clearing, and paraffin impregnation on an
automatic tissue processor. Cellular and subcellular morphology of
the resulting tissue sample is no different from that of tissue
samples fixed at room temperature in a routine procedure. However,
due to the low level of biomolecule cross-linking and modification,
availability for biomolecules to extraction and detection, as well
as quality and integrity of biomolecules, are significantly
improved.
[0046] For non cross-linking fixatives, also called coagulant
fixatives, such as alcohols, ketones, and other fixatives based on
alcohols and or ketones, as well as zinc-based fixatives, at cold
temperature, activity of most enzymes in the ischemic cascade is
suppressed, so autolysis is prevented during penetration of
fixatives into tissue specimens.
[0047] Extraneous physical forces, such as ultrasound, are applied
to overcome the lack of efficient penetration of fixative molecules
into tissue specimens at a cold temperature, i.e. in the range
between 0 degree C. to 25 degree C., and preferably 4 degree C. to
18 degree C., in order to shorten fixation time. Since ultrasound
generates moderate heat in the solution and tissue specimens in the
solution it irradiates, a cooling system is needed to keep the cold
temperature. The higher intensity the ultrasound irradiation is the
more powerful a cooling system must be. Ultrasound irradiation can
be continuous when a relative low intensity is used; or pulsate
when a relative high intensity is used. When fixative molecules
penetrate into tissue specimens under ultrasound irradiation, the
cooling system can be turned off, and solution and tissue
temperature is increased by ultrasound alone or in combination of
other heating mechanisms (e.g., microwave heating, infrared
heating, ohmic heating, and electronic heating). Ultrasound
facilitates penetration of fixatives into tissue specimens at cold
temperature; temperature is raised by ultrasound irradiation to
further facilitate cross-linking or coagulation of biomolecules in
tissue specimens.
[0048] In the example of formalin fixation, establishing a cold
penetration protocol can significantly slow down the biomolecule
changes as well as cross-linking formation during the penetration
stage. Once methylene glycol molecules are evenly driven into
tissue specimens, it will be possible for the succeeding
cross-linking reaction to be well controlled for tissues of
different type and size by a standard set of time and temperature.
The new procedure for the ultrasound-facilitated tissue fixation
may be as the following: 1) Immediately after removal from patients
tissue specimens are placed in cold fixative in (or optionally, in
a special US transparent container and transferred to) an
ultrasound device with a cooling system. The tissue specimens are
irradiated with ultrasound to speed up formalin penetration when
the cooling system is turned on to maintain cold temperature; 2)
Sound signal through and/or from tissue specimens is collected and
analyzed to monitor penetration (optional); 3) When penetration is
completed, the cooling system is turned off--continued ultrasound
irradiation, alone or in combination with other heating system(s),
brings tissue specimen temperature up to the cross-linking
temperature; 4) Sound signal through and/or from tissue specimen is
collected and analyzed to monitor cross-linking (optional); 5) When
cross-linking is completed, fixed tissue specimens can be subjected
directly to the ultrasound-facilitated processing procedure
(case-by-case workflow) or lower the temperature to 4 degree C. to
stop cross-linking process and wait for batched processing (routine
workflow).
[0049] In the example of fixation with formalin, penetration time
is dependent on tissue thickness. For tissues of thickness less
than 4 mm, cold penetration time should be controlled below 60 min
under ultrasound irradiation. To maintain low level of
cross-linking, fixation step should be performed only at a cold
temperature, with or without ultrasound irradiation. A cooling
system is used to superimpose on the ultrasound irradiation when
both cold temperature and ultrasound are necessary.
[0050] For tissues greater than 5 mm in thickness, fixative
penetration at a cold temperature (e.g., 4-15 degree C.) may take a
long time (longer than 2 hr) under ultrasound irradiation, and a
temperature slope is necessary to span the range of cold
penetration temperature to the cross-linking temperature (25-70 C).
The time required for temperature slope for tissues thicker than 5
mm is set to be in the range of 10 min to 6 hours. Ultrasound power
can be changed to adjust the heat delivered into the solution and
tissue specimens in it. The cross-linking temperature is set in the
range of 25 degree C. to 70 degree C., preferably in the range of
40 degree C. to 60 degree C., and further preferably in the range
of 45 degree C. to 55 degree C. The cross-linking time is set to be
in the range of 2 to 60 min, preferably in 5 to 15 min.
[0051] To control cross-linking/modification levels, chemical
reagents or an additional cooling step are used to prevent
prolonged cross-linking reactions which often leads to over
cross-linking. Since properties of different tissue types differ
greatly because of different structure and contents, time needed to
complete fixation and processing steps differ greatly from one
tissue type to another. Fixation and processing time are also
closely related to thickness of the tissue specimens to be
preserved as well as temperature of the preservation reagent. A
predetermined time means that duration of a fixation or processing
step is determined for each specific tissue type, thickness,
temperature of the preservation reagent, and ultrasound parameters.
According to our experiments, formalin fixation time is between 10
to 120 min for tissues of less than 3 mm in thickness at 4 to 15
degree C. under ultrasound irradiation. Fatty tissues, such as body
fat and mammary gland, need longer time to be fixed.
[0052] Ultrasound and temperature control can also be used in the
processing steps in tissue specimen preservation. Microcavitations
caused by ultrasound irradiation leads to instant alternative high
pressure and low pressure in the medium and tissue specimens
therein. This effect leads to emulsification and degassing in
tissues. Ultrasound also produces strong convection in the solution
under irradiation, leading to good solution circulation. No
stirring is necessary when ultrasound is applied. Ultrasound
simultaneously delivers heat into the medium solution and therefore
serves as a heating energy source.
[0053] Low pressure and optionally alternative low and high
pressures applied to the fixatives and other tissue preservation
reagents are helpful in improving tissue fixation and processing
procedures. Tissue preservation reagents include fixation reagents
(fixatives), dehydration reagents (alcohols, ketones, etc.),
clearing reagents (xylene, xylene substitute), and paraffin.
[0054] Typically, tissue specimens fixed in cross-linking fixatives
must undergo dehydration in alcohol to remove water. This invention
discloses using ultrasound to promote the dehydration step.
Temperature is an important factor in the dehydration process. The
higher temperature the dehydration step is performed, the shorter
time is needed, and vice versa. According to our experiments,
effect of ultrasound irradiation in the dehydration step is
maximized when proper temperature is reached. For fast completion
of the dehydration step, the dehydration solution is maintained at
0 degree C. to 25 degree C. below the boiling temperature of the
dehydration solution, and preferably 5 degree C. to 10 degree C.
below boiling temperature. For example, when 100% ethanol (boiling
point 78.4 degree C. under normal atmosphere pressure) is used as
dehydration solution, the optimal temperature is 50 to 78 degree C.
One other function of ultrasound irradiation in the dehydration
step is to maintain solution temperature, alone or combined with
other heating means. However, for the best morphological results
moderate temperature is required, although processing time may be
extended. Moderate temperature is in the range of 4 C to 50 degree
C. Ultrasound irradiation can significantly shorten dehydration
step at moderate temperature.
[0055] Clearing is a step in tissue processing in which the alcohol
is replaced by clearing agents such as xylene or xylene substitute.
Xylene and xylene substitute are inter-mixable with wax or
paraffin. So the clearing step is to facilitate the next wax
infiltration step. Clearing agents also function to remove fatty
components from tissue. This invention discloses using ultrasound
combined with temperature control to promote efficiency of the
clearing step. Ultrasound is applied in the clearing step at the
temperature range of 40 degree C. to 80 degree C., preferably 0
degree C. to 15 degree C. and further preferably 0 degree C. to 5
degree C. above the melting point of wax that is to be used in the
next infiltration step. Some researchers use 2-propanol as a
clearing agent. Since 2-propanol is poor in inter-mixing with wax,
it must be evaporated from wax either by heating and/or vacuum. In
this invention, we disclose that ultrasound is applied to evaporate
2-propanol from wax by its degassing and heating functions. The
preferred temperature range for ultrasound irradiation to evaporate
2-propanol is 0 degree C. to 15 degree C. under its boiling
temperature, which is 82.3 degree C. under normal atmosphere
pressure.
[0056] In the example of preparing tissue samples with coagulant
fixatives, such as alcohols, ketones, alcohol- or ketone-based
fixatives, and other non-cross-linking fixatives, initial cold
temperature is preferred for the fixatives. It is preferred that
tissue specimens are immediately placed in the refrigerated cold
fixative and kept at the cold temperature until ultrasound
irradiation is started, to promote biomolecule preservation. When
non-cross-linking fixative is used, no hard crust at tissue
periphery caused by cross-linking of proteins will form. Fixative
penetration at low temperature will function to slow down the
ischemic cascade or auto degradation in tissue specimens.
Ultrasound will be applied when the cooling system is turned off to
raise and keep the solution temperature, alone or in combination
with other heating means. According to our studies, coagulant
fixatives work most effectively at the temperature range of 0
degree C. to 20 degree C. below their boiling point, with or
without application of ultrasound. A temperature slope is often
necessary.
[0057] Since initial penetration of fixative solutions into tissue
specimens is performed at a refrigerated cold temperature where the
tissue structure is in a less fluidic state, ultrasound of low
frequency which causes strong cavitations can be used. Once tissue
specimens are fixed, they become harder and hence can endure
stronger cavitations. The US frequency range used in the disclosed
invention is 100 KHz to 5 MHz, preferably 200 KHz to 2 MHz, and
further preferably in the range of 0.4 MHz to 1.5 MHz. Ultrasound
power is in the range of 0.1 to 100 Watts of total output,
preferably between 5 to 60 Watts, further preferably between 10 to
30 Watts.
[0058] This invention also discloses a tissue sample preparation
device which comprises at least one ultrasound chamber, at least
one ultrasound generating and delivering device, a series of tissue
fixation and processing solutions, at least one cooling device
being coupled to the ultrasound chamber. Optional components
include, but not limited to, the following: specimen containers
holding a cold fixative, a refrigerated cabinet holding specimen
containers filled with a cold fixative, at least one heating system
coupled to ultrasound chamber, at least one sound signal collector,
at least one sound signal analyzer, and a central processing unit
(CPU).
[0059] The US reaction chambers are critical components. Besides
holding solutions and samples, they are responsible for delivering
the proper amount US energy to the solution in the chamber and to
the tissue samples. Our study showed that a uniform distribution of
US energy throughout the solution in the chamber is important for
consistent fixing and processing of tissues. The efficiency of
energy transmission from the transducer to the solution in the
chamber, a major factor of machine efficiency and reliability, is
determined by coupling efficiency. Our preferred US reaction
chamber will be a container made of stainless steel or another
metal alloy as a whole piece (a cup), with the transducer attached
to the outside face of the bottom of the container. Another
configuration of the ultrasound chamber include a metal, a plastic,
a glass, or a porcelain cup holding the fixative or processing
solution and a transducer housing removably submerged in the
solution in the cup, the transducer housing can be configured to
emit ultrasound wave from one side or from both sides. Function of
the transducer housing is to seal the piezoelectric transducer from
preservation reagents submerging the transducer. To emit ultrasound
from one side of the transducer housing, the transducer is attached
to one flat side of the housing by a glue while the other face of
the transducer is unbounded. To emit ultrasound from both sides of
the transducer housing, the transducer is either attached to both
flat sides of the housing by a glue, or freely submerged in an
inert liquid in the housing. In this case, ultrasound is
transmitted through the inert liquid and both flat sides of the
housing to the tissue preservation reagents. Tissue specimens can
be placed on both sides of the transducer housing if ultrasound is
emitted from both sides of the housing, doubling the number of
tissue specimens to be preserved.
[0060] In one embodiment of formalin fixation step, the low
temperature phase (0 degree C. to 15 degree C.) and the high
temperature phase (35-75 degree C.) can either be performed in a
same US chamber or be performed in two different US chambers each
holding a fixative solution of relevant preferred temperature.
[0061] In one embodiment, vacuum can be applied to the solution and
the tissue specimens in the US chamber. Vacuum can be generated by
a separate vacuum pump. A moderate vacuum can also be generated by
a piston-pipe structure built into the US chambers.
[0062] Conventional laboratory cooling systems include
refrigerators, freezers, or even ice and dry ice. We tested the
cooling effects of a 4 degree C. refrigerator, an ice-water bath,
and a -20 degree C. freezer, to cool down water of 250 ml in a US
chamber with US irradiation at a fixed 50 W of electronic power
output. Temperature eventually could reach a stable level but could
not be controlled by the operator. We disclose a temperature
adjustable cooling/heating system capable of being coupled directly
to the US chambers. It will comprise a refrigerated circulating
bath (adjustable temperature range -20 degree to 60 degree C.) and
a circulating temperature-coupling device, such as a copper pipe
coil, connected to the refrigerated circulating bath by rubber
tubing, or a cooling jacket. Circulating liquids can be water,
ethanol, Freon. Heating can also be achieved by wrapping a heating
pad around the US chamber.
[0063] It is important to insure that fixation and processing steps
are adequately accomplished, especially in fast procedures.
Therefore, a quality control system is necessary. When ultrasound
is applied to facilitate fixation and processing, reflected and/or
transmitted ultrasonic signals from tissue samples being fixed and
processed can provide information to indicate the progress of each
step, and an acoustic monitoring system with modest complexity can
be established.
EXAMPLES
Example 1
[0064] We treated lysozyme and BSA with NBF for 60 min at various
temperatures and immediately loaded the cross-linked products onto
SDS gels for separation. For both lysozyme and BSA, cross-linking
level gradually increased in the temperature range between 0 degree
C. and 40 degree C. As revealed by the SDS PAGE, there is a
significant increase in size of oligomers (lysozyme) or in gel
mobility (BSA) at 50 degree C., which peaks at around 60 degree C.
The inventors noticed that large amount of aggregates began to form
when incubating lysozyme with NBF at a temperature greater than 60
degree C. These aggregates were highly intermolecularly
cross-linked lysozyme molecules, and could not be separated on SDS
PAGE. No aggregates were observed with BSA in NBF at high
temperatures, possibly due to the fact that cross-linking in BSA
was mostly intramolecular.
[0065] In the case of lysozyme, there was a gradual acceleration of
cross-linking speed at temperature of 0-20 degree C. and leveled at
the range of 20-40 degree C. which then followed by abrupt speed
increase at temperatures 50 degree C. and up. While in the case of
BSA, cross-linking gradually accelerated at temperatures from 0
degree C. to 30 degree C. and abruptly accelerated at temperatures
40 degree C. Experiments with these two typical proteins showed
that speed of cross-linking formation in formalin fixation
accelerated with increase in temperature, and speed acceleration
may be classified into two modes according to incubation
temperature: 1) slow acceleration which happens at 0 degree C.-40
degree C., and, 2) fast acceleration which happens at 40-100 degree
C. With both proteins, we speculate that temperature between 50
degree C. and 60 degree C. is an important range at which
cross-linking happens at high but controllable speed. At
temperatures higher than 60 degree C., insoluble aggregations
caused by intermolecular cross-linking began to form in large
amount, as revealed by the experiment with lysozyme.
Example 2
[0066] Significant cross-linking occurs after 10-minute incubation
at 50 degree C. In another experiment, we tested cross-linking
formation for various proteins when incubated in formalin at 4
degree C., RT, and 50 degree C. for 10 min, in comparison with
incubation with formalin at RT overnight. As shown in FIG. 5,
cross-linking at 4 degree C. was the slowest for all the proteins
tested. And there is a big increase in levels of cross-linking
after incubation at 50 degree C. for 10 min, almost comparable to
the cross-linking levels after overnight incubation at room
temperature. For all proteins except BSA, protein precipitations
formed when incubated with formalin at 50 degree C. for 10 min and
at RT overnight. This experiment indicated that for many proteins
incubation with formalin at 50.degree. C. for 10 minutes lead to
significant cross-linking formation. Similar results were also
obtained for 5-minute incubations (data not shown).
Example 3
[0067] Tissue specimens of 1-4 mm thick were held in tissue
cassettes and submerged in a cross-linking fixative (e.g. formalin,
4-25 degree C.) until ultrasound irradiation. Ultrasound is applied
to bring the fixative temperature up to 50-80 degree C. alone or in
combination with other heating means. The temperature was
maintained for 5 to 20 min.
[0068] The tissue specimens were then submerged in a dehydration
agent, e.g., 100% ethanol. Temperature of the dehydration agent was
brought up to 50-75 degree C. by ultrasound irradiation alone or in
combination with another heating means. The temperature was
maintained by ultrasound irradiation alone or in combination of
another heating means for 5-30 min. This step was repeated at least
once.
[0069] The tissue specimens were then submerged in a clearing
agent, e.g., xylene or xylene substitute. Temperature of the
clearing agent was brought up to 50-75 degree C. by ultrasound
irradiation alone or in combination with another heating means. The
temperature was maintained by ultrasound irradiation alone or in
combination of another heating means for 5-30 min. This step was
repeated at least once.
[0070] The tissue specimens were then submerged in melted wax or
paraffin at a temperature range of 55-70 degree C. Ultrasound
irradiation was applied to melted wax or paraffin for 5-30 min.
Temperature of the wax or paraffin was maintained at 60-75 degree
C. by ultrasound irradiation alone or in combination with another
heating means.
Example 4
[0071] Tissue specimens of 1-4 mm in thickness were held in tissue
cassettes and submerged in a non-cross-linking fixative (e.g., 100%
ethanol, acetone, or a mixture of 40% isopropyl alcohol, 40%
acetone, 20% polyethylene glycol (average molecular weight 300) and
1% dimethyl sulfoxide (DMSO)) at the temperature range of 4-25
degree C. Ultrasound was applied to bring temperature to the range
of 50-70 degree C. alone or in combination with another heating
means. The temperature was maintained for 5 to 20 min. Dehydration,
clearing, and wax infiltration steps were performed as described in
Example 6.
Example 5
[0072] Tissue specimens of 3 mm or thinner were held in tissue
cassettes and submerged in NBF at temperature of 50-70 degree C.
Ultrasound is applied for 5-15 minutes and temperature of NBF is
maintained by ultrasound irradiation alone or in combination with
another heating means. Dehydration, clearing, and wax infiltration
steps were performed as described in Example 6.
Example 6
[0073] Fresh tissue specimens of 5 mm or thicker were submerged in
NBF at a temperature range of 4-10 degree C. Ultrasound was applied
to NBF. To maintain temperature within 4-10 degree C. a cooling
system was applied to cool down NBF. Ultrasound irradiation lasted
for at least 30 minutes.
[0074] The cooling system was turned off. NBF temperature was
raised by continued ultrasound irradiation until temperature
reached the range of 55-80 degree. Temperature was maintained by
ultrasound irradiation alone or in combination with a heating pad
for 10-30 minutes.
Example 7
[0075] We fixed fresh bovine kidney, liver, and pancreas tissues (3
mm.times.5 mm.times.5 mm) in formalin overnight at 4 degree C. or
room temperature and processed the fixed tissues on an automatic
tissue processor. Tissues fixed overnight at 4 C still appeared
fresh while tissues fixed overnight at RT were grayish. 3 micron
sections from the FFPE paraffin blocks were stained by H&E and
viewed under a microscope. Cell morphology in tissue samples fixed
at 4 degree C. generally had brighter and sharper edge as well as
better sub-cellular details. Low temperature fixation greatly
reduced vacuolization commonly existing in tissues fixed by the
routine fixation procedure. Red blood cell lysis was also reduced
in tissues fixed in cold formalin. The most striking difference
between the two fixation methods is demonstrated in pancreas
samples. Pancreas tissues fixed in RT formalin overnight showed
overt destruction in cellular structures in the islets. Number of
nuclei per field and nuclear size are calculated to analyze whether
there is any cellular or intracellular shrinkage due to low
temperature. The quantitative counting results showed no detectable
change due to the low temperature; contrary to a previous study
with liver tissue that formalin fixation at 4 degree C. overnight
resulted in tissue shrinkage [Fox 1987]. Experiment with the human
ovary cancer tissue specimen also showed that fixation at 4 degree
C. produced better morphological features than those by the
conventional room temperature method.
Example 8
[0076] We studied US effect in promoting diffusion of formalin
molecules into tissue specimens at a refrigerated temperature using
human ovary and liver tissues. Fixation at 4 degree C. for 18 hr
generated better morphological features than fixation at 8 degree
C. for 5 min under US, which is better than the conventional RT
fixation for 18 hr.
[0077] Human liver tissue (3 mm thickness) was fixed in 4 degree C.
formalin with US irradiation for 30 minutes. As a control, the same
liver tissue of similar size was fixed in 4 degree C. formalin for
30 minutes without US. The liver tissue fixed with US irradiation
showed good morphological features. While the liver tissue fixed
without US irradiation demonstrated severe loss of red blood cell
integrity and subcellular structure details. Since the succeeding
processing steps were done through regular alcohol dehydration,
xylene clearing and paraffin embedding, the tissue fixed in 4
degree C. formalin for 30 min without US produced alcohol fixation
morphology while the tissue fixed in 4 degree C. formalin for 30
min with US produced excellent formalin fixation morphology.
Example 9
[0078] Cow kidney tissue (3 mm thickness) was fixed in 4 degree C.
formalin for 30 min with US irradiation (US-LT-FFPE) or at RT
overnight (FFPE), and then processed on automatic tissue processor.
IHC was carried out for vimentin and cytokeratin without the
antigen retrieval (AR) process. In both IHC assays, the sample
fixed with US irradiation produced better results than the routine
FFPE sample. The percentage of IHC stain positive area in
US-LT-FFPE indicates that more antigen epitopes (antigenic
determinants) are preserved than routine FFPE methods. Protein
extracted from tissue specimens also showed that US facilitated
fixation at 4 degree C. resulted in stronger signals for both
vimentin and cytokeratin than routine FFPE for Western blot. This
data indicates that antigens in the US-LT-FFPE tissue sample are
modified to a lesser degree and more available for IHC detection
and extraction than the routine counterpart because of reduced
cross-linking levels.
Example 10
[0079] To study if US can facilitate formalin fixation of large
tissues, we fixed a human autopsy whole prostate specimen in
formalin with US irradiation at 10 degree C. for 16 h and at 25
degree C. for 2 h. As a control, a surgically removed patient's
whole prostate specimen was fixed in formalin at RT. After the 18-h
fixation, the prostate specimen fixed by the US method still looked
fresh with blood, fat, and capsule tissues maintaining their
original appearances. The prostate specimen that was fixed
overnight by the conventional method looked gray. We roughly
compared hardness of both prostates by touching them with our
fingers. The prostate fixed by the US method appeared to be harder
than the one fixed by the conventional method.
[0080] Both prostate specimens were subjected to an MRI examination
after overnight fixation. The prostate fixed by the US method
generated darker and more even images on every plane selected in
both the peripheral and center regions while the conventionally
fixed prostate generated uneven images with scattered bright spots
in the center region. The dark areas in an MRI image are correlated
with the density of the tissue specimen [Hennig 1986].
Cross-linking caused by formaldehyde creates a density change in
tissue specimens that can be revealed by MRI.
Example 11
[0081] We have designed a series of direct forward biochemistry
experiments to compare various chemical reagents such as formalin,
RNA Later, methanol/alcohol, as well as heating conditions (boiling
or autoclaving) in inhibiting the RNase bioactivity. The study
results indicate that formalin is the best RNase inhibitor. We also
compare the inhibition ability of formalin at room temperature and
4 degree C. No difference was found under this comparison study.
This result suggests that instant formalin inhibition of the RNase
activity at 4 degree C. is not dependent on slow cross-link
reaction. This is the primary data on free solution reaction,
however, the preservation of RNA by cold formalin at tissue level
should be further studied. According to the cell protein and RNase
experimental results, we suggest that the low temperature formalin
fixation can achieve the similar enzyme inhibition ability and it
can preserve the biomolecules with low modification (low level
cross-linking).
Example 12
[0082] To make a rough comparison on proteins extractable from
samples fixed at 4 degree C. and RT, we did a time course study
measuring 2 specific proteins with Western blot using cultured
cells. Diffusion time of formalin molecules into cultured cells can
be neglected, in this case incubation time can better represent the
cross-linking time. Aliquots of freshly grown breast cancer T47D
cells were treated with neutral buffered formalin (NBF) at 4 degree
C. or RT for different lengths of time. Proteins in cell lysates
were separated by SDS PAGE and then transferred to a nitrocellulose
membrane which was probed with pooled monoclonal antibodies against
HER-2 and ER, a membrane protein and a nuclear protein
respectively. Coomassie blue stained SDS gel showed that cells
fixed at 4 degree C. generated more proteins lysates throughout
fixation time 0.5 to 18 hr) than cells fixed at RT. Western blot
showed that at 4 degree C. fixation conditions similar signal
strengths for HER-2 and ER proteins were produced for fixation
times between 0.5 to 4 hr, while at RT only the 0.5 hr fixation
time produced readable signals for both proteins. Strikingly, we
found that, in terms of protein extractability, RT fixation for 0.5
hr is roughly equivalent to 4 degree C. fixation for 18 hr.
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